Abstract

In the last few decades, evidence has been accumulating for a role for xanthine
oxidoreductase (XOR)-generated toxic reactive oxygen species (ROS) in a variety of
pathological conditions that affect different organ systems. This enzyme in mammals
exists in two inter-convertible forms: xanthine dehydrogenase (XDH) (the predominant
intracellular form under physiological conditions) and xanthine oxidase (XO). A
combination of XO and its oxidizable substrate xanthine (X) (or hypoxanthine (HX)) is
widely used as a model to produce ROS and to study their effects in a variety of cell
culture studies. However, the effect of the combination of XOR and the reduced
nicotinamide adenine dinucleotide (NADH) in cell cultures is much less studied. NADH
is another oxidizable substrate for XOR that binds to a different site on the enzyme from
that of X binding.
The aim of this project was to investigate some aspects of the in vitro toxicity of XOR,
which might provide more insights into its in vivo toxicity. The main investigation was
a comparison between the well studied X / XO and the much less studied NADH / XO
toxicity models. Also, secondary studies were undertaken to investigate those aspects of
X / XO toxicity where there are uncertainties about them.
These studies were performed using primary cell cultures. Cell cultures are now widely
used to study different diseases, and although they have their drawbacks, they have their
advantages over the in vivo studies. For this project, primary cultures of cerebellar
granule neurons (CGNs) were used. In the beginning, some problems were encountered
with CGNs. The main problem was the immediate damage induced to the neurons
(including those in the control groups) at the intervention/experiments day (i.e. day 8 or
9 after plating) by manipulating the cultures (i.e. aspirating the culture medium, adding
treatment and control vehicles, and adding the restoration medium).
After several months of investigation, it was serendipitously discovered that the
immediate damage seen in the neurons (including those in the control groups) when
they are manipulated at the experiments/intervention day was due to glutamate
excitotoxicity (through activating its N-methyl-D-aspartate (NMDA) receptors). The
source of glutamate was the fresh serum which is present at 10% V/V in the fresh
culture medium that is added to the cultures at that day. After solving this problem, it
was possible to conduct reliable experiments to investigate XO toxicity models.
Regarding investigating XO toxicity, it was found that both of the X / XO and NADH /
XO combinations were toxic to cultures of CGNs. However, the concentration of
NADH needed to cause the toxicity was much higher than that of the other substrate, X,
which is in agreement with previous cell-free experiments that showed that NADH is a
much weaker substrate than X for the bovine milk XO used here. Blocking the site of X
binding on XO prevented X / XO toxicity, but did not prevent NADH / XO toxicity. On
the other hand, blocking the site of NADH binding prevented both X / XO and NADH
/XO toxicities. Another difference between the two systems was that deactivating either
superoxide or hydrogen peroxide (both are ROS) generated by XO prevented NADH /
XO toxicity, whereas although deactivating hydrogen peroxide prevented X / XO
toxicity, deactivating superoxide generated from this combination did not. In the NADH
/ XO system, an extracellular metal contaminant (likely contaminating XO
powder/preparation) seemed to be involved in the toxicity. The two toxicity models
were similar in the mediation of toxicity by intracellular iron ion. In X / XO toxicity,
although superoxide generated extracellularly from the combination has no role in the
toxicity, intracellularly produced superoxide seemed to play a role.
Conclusions:
1. Culturing/experimental conditions have been optimised for viability studies in
CGNs cultures.
2. The combination of NADH and XO induces damage to CGNs, where although
blocking the NADH binding site prevents this damage, blocking the X binding
site does not. It is feasible that the oxidation of NADH by some forms of XOR
(other than the one used here) that are known to be very efficient in oxidizing
NADH might produce in vivo toxicity.
3. A possibility raised by this study is that a metal (like the metal contaminant
proposed to play a role in NADH / XO toxicity in this study) might contribute to
XOR toxicity in vivo.
4. Intracellular superoxide often mediates XOR toxicity.
5. The results add support to many previous studies which suggested that
intracellular hydroxyl radical (or a similar species) is involved in XOR toxicity.